Techniques for Preparing a Canine Vaginal Smear

Materials Required to Obtain and Process Vaginal Smears

methanol or commercial spray fixative for cytology (can also use ethanol)

staining solution: a commonly used product is Diff-Quik (American Scientific Products), but almost any stain used for blood smears will work well (e.g. Giemsa or Wrights stain)

Obtaining the Sample

The objective is to obtain a sample of epithelial cells from the vagina, and one should avoid sampling from the vestibule (i.e. just inside the vulva). Even in small dogs, the swab should be inserted several inches past the vulva; in large breed dogs (e.g. German Shepard bitch), a majority of the 6-inch swab can be inserted. Bitches in proestrus or estrus rarely object to this procedure, although some restraint may be required to prevent "squirming".

Part the lips of the vulva and gently insert a swab at a relatively steep angle. Some people suggest using a speculum (e.g. the cone from an otoscope) and some like to moisten the swab with saline prior to use, but neither of these is critical. After 1-2 inches of the swab have been inserted, the angle of insertion can be altered to roughly 45 degrees and insertion continued.

When the swab is fully inserted, rotate the end through 2-3 revolutions, which will allow the cotton tip to pick up an adequate load of cells. The swab can then be gently withdrawn. The images above show this procedure courtesy of Ruby the Alaskan Husky.

Preparing and Staining the Smear

Prepare the smear immediately after withdrawal of the swab by rolling (not sliding or rubbing) the cotton tip along the length of a glass microscope slide. Generally, two parallel tracks can be rolled on a single slide.

As soon as the smear is prepared, dip it 5 to 10 times in a container of methanol, or fix by use of a spray fixative. It is best to fix expeditiously, but allowing the slide to dry fully and remain unfixed for up to a few hours is generally not a problem. After fixation, the slide can be stored for prolonged periods of time, although typically, it is stained without delay.

Diff-Quik is a very convenient stain for use with vaginal smears. It consists of two solutions: an eosinophilic (red) solution and a basophilic (blue) stain. It is best to have a small container of each component and replace the stains with some regularity, as they will become contaminated with vaginal cells after staining a number of slides. To stain the smear, dip the slide in and out of the red stain 5 to 10 times, the in and out of the blue stain 5 to 10 times.

There is no need to rinse the slide between red and blue, but do not dip first into the blue, then into the red, or the red will rapidly become blue! If the staining appears too light or too dark, adjust the number of dips accordingly. A number of other stains can be substituted for Diff-Quik.

After dipping in the blue stain, rinse the slide in tap water and it's ready to examine. Examine while still wet or dry the slide and apply a coverslip.